- Email: [email protected]
Frozen-section biopsy analysis for acute invasive fungal rhinosinusitis
Otolaryngology–Head and Neck Surgery (2007) 136, 714-719
ORIGINAL RESEARCH
Frozen-section biopsy analysis for acute invasive fungal rhinosinusitis Murtaza T. Ghadiali, MD, Nathan A. Deckard, BS, Uzma Farooq, MD, Frank Astor, MD, Phillip Robinson, MD, and Roy R. Casiano, MD, Miami, FL OBJECTIVE: To evaluate sensitivity and specificity of frozensection biopsy in the diagnosis of acute invasive fungal rhinosinusitis (AIFRS). STUDY DESIGN AND SETTING: Retrospective chart review of all patients treated for AIFRS at the University of Miami between 1993 and 2005. Twenty patients with the clinical diagnosis of AIFRS were identified. Histopathological data were collected to evaluate the use of frozen-section biopsy to diagnose the disease. RESULTS: Permanent pathology sections were positive for AIFRS in all specimens. Frozen-section biopsies were positive for fungal features in all but five cases (four with Mucor and one with Aspergillus). High positive predictive values were found for both fungal types and for both sets of patients (bedside and intraoperative). CONCLUSION: Frozen-section biopsy is a useful tool for rapid and effective diagnosis in patients with suspected AIFRS. A positive frozen-section result is highly predictive of invasive disease and merits prompt surgical intervention. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
A
cute invasive fungal rhinosinusitis (AIFRS) is an uncommon, opportunistic infection seen primarily in diabetic and immunocompromised patients. The infections are predominantly by species from the order Mucorales (including Absidia, Mucor, Rhizomucor, and Rhizopus) and by Aspergillus. The spores of these fungi are ubiquitous in the environment. As a result, exposure is common in all population groups, although progression to invasion and infection is usually avoided with an intact immune system.1,2
From the University of Miami/Jackson Memorial Hospital, Department of Otolaryngology/Head and Neck Surgery (Drs Ghadiali, Astor and Casiano), Department of Pathology (Drs Farooq and Robinson) and the University of Miami Miller School of Medicine (N. Deckard). Disclosure: Dr Caslano: Gyrus ENT-royalties, consultant; Medtronics. Presented at the American Academy of Otolaryngology–Head and Neck Surgery, Toronto, Canada, September 19, 2006.
Mucormycosis in particular has been associated primarily with diabetes mellitus patients in ketoacidosis.2 Other patient groups commonly affected by AIFRS include those with neutropenia, immunological disorders, chemotherapy, and transplant recipients. The progression of the disease depends on the severity of immunosuppression; the greater the immunocompromise, the more rapid the progression.1 AIFRS first affects the turbinates and sinuses resulting in an acute sinusitis. The infection can then spread to contiguous structures such as the palate, the orbit, and the brain leading to cranial nerve palsies, invasion of the carotid artery, and mental obtundation, thus making early diagnosis key before such extensive invasion occurs.1 Mucor has a predilection for the internal elastic lamina of the arterial blood vessels and spreads by angioinvasion. This causes tissue necrosis as the arterial blood flow is occluded, resulting in infarction and the black eschars that are pathognomonic for the disease.2,3 Furthermore, it has been shown that perineural invasion is also an integral aspect of the disease process of Mucor and can be used to identify the invasive nature of this fungus on histological examination.3 Aspergillus has a similar picture in that, in its invasive form, its hyphae may also invade the vasculature and spread by angioinvasion. It may also spread directly across tissue planes.4 Although sinus CT is the preferred imaging modality to evaluate for signs of invasion by the disease, bony destruction is often seen only late in the course of the disease after soft-tissue necrosis has already occurred. AIFRS can present without any evidence of invasion on an essentially Reprint requests: Murtaza T. Ghadiali, MD, University of Miami Hospital and Clinics, c/o Dr Roy R. Casiano, 1475 NW 12th Avenue, Room 4025, Miami, FL 33136. E-mail address: [email protected].
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.01.002
Ghadiali et al
Frozen-section biopsy analysis for acute invasive . . .
normal CT scan.5 Therefore, any clinical indication of an invasive process warrants a biopsy at the bedside or in the operating room to confirm a tissue diagnosis of invasive fungal disease. Frozen-sections at the time of the original biopsy have been recommended to obtain the earlier diagnosis so that medical treatment can begin as soon as possible in the event of a positive diagnosis.2,6-9 Past studies have found frozen-section to be useful in diagnosis and even during surgical debridement.6-8 However, there is some evidence that its usefulness is limited.9 There is a paucity of reports in the literature that have statistically evaluated frozen-section as a diagnostic tool in the evaluation of AIFRS patients. In this study, we have conducted a retrospective review of patients’ charts who have undergone surgery for this diagnosis, looking specifically at the predictive value of frozen-section to diagnose the condition.
METHODS The medical records from the University of Miami/Jackson Memorial Hospital were reviewed from 1993 to 2005 for all cases of acute invasive fungal rhinosinusitis after approval was obtained from the institutional review board. All cases of invasive Mucor and Aspergillus infections proven by permanent section that also had frozen-sections performed were included in this retrospective chart review. Data on age, sex, comorbidities, and clinical presentation were collected from the charts. Specific comorbidities that were noted were those that cause an immunocompromised state, such as diabetes mellitus, solid-organ transplant, immune system disorders, neutropenia, chemotherapy, and HIV/AIDS. Frozen-section slides were stained with hematoxylin and eosin. Permanent sections were stained with Gomori methanamine silver and immunoperoxidase antibody stains. All slides were analyzed under the microscope at 500⫻ by the University of Miami/Jackson Memorial Hospital Department of Pathology. Mucor was identified by its broad, thin-walled, and sparsely septated hyphae. The hyphae are typically nonparallel, branch irregularly, and are irregular in size (Fig 1). Aspergillus was identified by its septated and regularly branched hyphae of uniform diameter. The branching is characteristically at a 45° angle (Fig 2). Sinus tissue was sent to the microbiology laboratory and cultured using Inhibitory Mold Agar and Sabouraud Dextrose Agar per laboratory protocol. Histopathological examination reports (including frozen and permanent section specimens used to diagnose patients before treatment, during intraoperative debridement, and postoperatively) as well as microbiological culture results were obtained and analyzed.
STATISTICAL ANALYSIS A generalized estimating equation (GEE) approach10 with a logit link and a working independence correlation structure
715
was used when estimating 95% confidence intervals for sensitivity and negative predictive value (NPV) to take into account potential correlations between outcomes in samples from several sites in the same individuals. The GEE technique adjusts the standard errors to provide appropriate asymptotic inferences in repeated measures data. For specificity and positive predictive value (PPV), exact Blyth 95% confidence intervals11 were calculated because these quantities were 1, meaning that there would have been numerical problems with GEE estimates. Assuming positive correlations between the outcomes in samples on the same individual, the true confidence intervals would probably be wider. Differences in the sensitivity and NPV between different fungal species as well as intraoperative and other patients were tested for significance by entering a group main effect into the intercept-only GEE model and assessing the P value from the Wald test. These models also yielded 95% confidence intervals for sensitivity and negative predictive value in these different groups. Because specificity and PPV were both 1 in all groups, we could not perform any tests for differences in these quantities between different fungal species and intraoperative and other patients. However, exact Blyth 95% confidence intervals for these quantities were calculated in these subgroups. All tests were 2 sided, and alpha was set at 0.05. All analyses were performed by using SAS/STAT software, version 9.1.3 of the SAS System for Windows (SAS Institute Inc, Cary, NC).
RESULTS A total of 20 patients with the clinical diagnosis confirmed by histopathology on permanent section that also had frozensections performed were identified from a chart review from 1993 to 2005. The age range was from 8 to 76 years, with an average age of 44 years. There were a total of 9 males and 11 females (Table 1). The study included 11 patients with a diagnosis of invasive mucormycosis and 9 patients with invasive aspergillosis. Of these, the most common predisposing illness was diabetes mellitus, occurring in 11 patients (55%). Solidorgan transplant–related immunocompromise was found in five patients (25%), HIV/AIDS was a diagnosis in five patients (25%), and leukemia was a diagnosis in one patient (5%). Two patients had no identified immunocompromised states that would predispose them to invasive fungal disease. Some of the patients in the study had multiple comorbidities. The most common presenting signs and symptoms included facial pain and swelling in 9 of 20 patients (45%), proptosis/visual changes in 8 of 20 patients (40%) and headaches in 4 of 20 patients (20%). Other presenting symptoms included sinusitis, bloody nasal drainage/epistaxis, necrotic hard palate, and V2 numbness. Among the 20 patients, a total of 44 samples were collected that were processed for both frozen and permanent
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Otolaryngology–Head and Neck Surgery, Vol 136, No 5, May 2007
Table 1 Frozen-section Patient
Age
Sex
Comorbidities
SSx
Dx
1 2 3
50 61 76
F M F
HIV/AIDS, DM KTSPL, DM DM
Vision loss, proptosis Facial pain Facial pain, swelling
Mucor Mucor Aspergillus
4 5 6
63 35 17
M F F
DM LivTSPL DM
Facial pain, HA Necrotic palate Facial pain, swelling
Mucor Mucor Mucor
7 8 9 10 11
49 35 31 59 72
F F M M F
LuTSPL, DM HIV/AIDS HIV/AIDS HIV/AIDS DM
Facial pain, swelling Vision loss, proptosis Facial swelling, proptosis Blindness, HA Facial swelling, proptosis
Aspergillus Aspergillus Aspergillus Aspergillus Mucor
12 13 14 15 16 17
43 40 8 25 38 48
M M F M F F
None Acute Leukemia KTSPL, DM N/A DM DM
Sphenoid sinusitis Bilateral sinusitis Facial pain, swelling Nasal mass, sinusitis Vision loss Facial swelling
Aspergillus Mucor Mucor Aspergillus Mucor Mucor
18 19 20
21 62 49
F M M
HIV/AIDS DM LivTSPL
Vision loss, epistaxis HA, EOM palsy HA, V2 numbness
Aspergillus Aspergillus Mucor
Frozensection
Permanent section
Positive ⫻1 Positive ⫻1 Positive ⫻1 Negative ⫻7 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻7 Negative ⫻1 Negative ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻2
Positive ⫻1 Positive ⫻1 Positive ⫻1 Negative ⫻7 Positive ⫻1 Positive ⫻1 Positive ⫻2 Positive ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻7 Positive ⫻1 Negative ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻2
DM, diabetes mellitus; KTSPL, kidney transplant; LivTSPL, liver transplant; LuTSPL, lung transplant; HA, headache; EOM, extraocular muscle *Italicized entries denote specimens obtained during intraoperative debridement.
section. All 20 cases were positive on permanent section for invasive fungal disease. For patients 18 and 19, the biopsies sent for both frozen-section and permanent section were returned as negative as noted in Table 1. However, separate biopsies were sent for permanent section only, which confirmed the diagnosis of AIFRS. Frozen-section results were positive for fungal features in all but 5 of the 44 samples collected. Four biopsies with Mucor and one with Aspergillus were falsely negative. Using permanent section as the gold standard, overall sensitivity for frozen-section was 0.84 (95% confidence interval [CI], 0.67-0.93), specificity was 1 (95% CI, 0.75-1), NPV was 0.72 (95% CI, 0.37-0.92), and PPV was 1 (95% CI, 0.87-1). For Mucor alone, sensitivity was 0.83 (95% CI, 0.64-0.93), specificity was 1 (95% CI, 0.39-1), NPV was 0.5 (95% CI, 0.18-0.82), and PPV was 1 (95% CI, 0.83-1). For Aspergillus alone, sensitivity was 0.86 (95% CI, 0.42-0.98), specificity was 1 (95% CI, 0.66-1), NPV was 0.90 (95% CI, 0.42-0.99), and PPV was 1 (95% CI, 0.54-1). No statistical difference was found for the sensitivity or NPV of frozen-section biopsy between the two fungal groups (P ⫽ 0.88 and 0.14, respectively). However, given the large confidence intervals for these figures, the study may not have been adequately powered.
Six of the 20 patients had frozen-sections performed intraoperatively to guide the surgical debridement. A total of 30 slides were performed on these six patients alone. Within this group, the sensitivity for frozen-section was 0.84 (95% CI, 0.62-0.94), specificity was 1 (95% CI, 0.72-1), NPV was 0.79 (95% CI, 0.35-0.96), and PPV was 1 (95% CI, 0.79-1). For the nonintraoperative group, the sensitivity for frozen-section was 0.83 (95% CI, 0.52-0.96) and NPV was 0.5 (95% CI, 0.120.88). No statistical difference was found for the sensitivity or NPV of frozen-section biopsy between the two groups, intraoperative and nonintraoperative patients (P ⫽ 0.95 and 0.35, respectively). However, again, the large confidence intervals for these data may indicate an inadequate power for this study (Table 2). Using permanent section as the gold standard, overall culture results were positive in only 11 of the 20 cases. Overall sensitivity for cultures was 0.55 (95% CI, 0.340.74). For Mucor alone, sensitivity was 0.54 (95% CI, 0.29-0.78). For Aspergillus alone, sensitivity was 0.55 (95% CI, 0.27-0.80). Specificity was not calculated for cultures because there were neither true negatives nor false positives in our population (Table 3).
Ghadiali et al
Frozen-section biopsy analysis for acute invasive . . .
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Table 2 Intraoperative frozen-section Patient
# sent intraop
FS Diagnosis
#TP
#FP
#TN
#FN
PS diagnosis
3 6 11 14 17 20 Totals:
8 4 9 2 3 4 30
Aspergillus Mucor Mucor Mucor Mucor Mucor
1 2 7 2 2 2 16
0 0 0 0 0 0 0
7 0 1 0 1 2 11
0 2 1 0 0 0 3
Aspergillus Mucor Mucor Mucor Mucor Mucor
DISCUSSION Acute invasive fungal rhinosinusitis is an uncommon infection that occurs almost exclusively in immunocompromised patients. An early diagnosis allows the prompt institution of medical and surgical treatment in order to avoid the high morbidity and mortality associated with the progression of the disease.1,2,12,13 The problem that clinicians face in practice is finding a rapid and reliable means by which to diagnose the infection. The gold standard of diagnosis is pathological examination on permanent sections.1-3 However, permanent section can be a lengthy and time-consuming means to obtain the diagnosis, delaying the appropriate treatment and increasing the morbidity and mortality of the disease. The current literature available on the accuracy and utility of frozensection in the diagnosis of this disease has been sparse. The literature has thus far been limited in scope to case reports and one small retrospective study that described the use of frozen-section in diagnosis but included only Mucor pa-
Table 3 Culture results Patient
PS diagnosis
Culture diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Mucor Mucor Aspergillus Mucor Mucor Mucor Aspergillus Aspergillus Aspergillus Aspergillus Mucor Aspergillus Mucor Mucor Aspergillus Mucor Mucor Aspergillus Aspergillus Mucor
Negative Rhizopus A. flavus Rhizopus Negative Negative A. fumigatus A. fumigatus A. fumigatus A. fumigatus Rhizopus Negative Negative Rhizopus Negative Rhizopus Negative Negative Negative Rhizopus
tients and did not convey any numerical data for the sensitivity and specificity of the procedure.6-9 This study differs from the existing literature in that our patient population contains a total of 44 frozen-section specimens, includes both Mucor and Aspergillus in the analysis, and provides statistical analyses to support its conclusions. In this retrospective study, a total of 44 frozen-sections were obtained from 20 patients. The utility of frozen-section biopsy was evaluated on a site-specific basis to compare how well it performs as compared with permanent section. Only five were found to be false negatives based on the final permanent section results of the same specimens, showing a sensitivity of 84% and an NPV of 72%. In addition, the narrow confidence interval of the PPV shows that frozensection biopsy is highly predictive of invasive disease. Therefore, frozen-section diagnosis was found to be a useful tool for the early diagnosis of AIFRS in the event that the patients’ clinical appearance alone does not indicate invasive disease to the examiner. Furthermore, no statistical difference was found for the sensitivity or the NPV of frozen-section between the two most common fungal species to cause AIFRS, Mucor and Aspergillus. Frozen-section does not just provide an early diagnosis in the clinical setting. It can also provide useful information for frozen-section– guided intraoperative and postoperative debridement in the operating room. Six of the patients in our review used this method as a part of their surgical treatment. A total of 30 slides were obtained within this group alone. Using the pathological reports, it was found that intraoperative frozen-section results for sensitivity and NPV were not statistically significant from the nonintraoperative patients. This is useful to the surgeon in guiding his/her procedure. By knowing that frozen-section is a reliable tool, it can be used to prevent more extensive surgical debridement by providing intraoperative negative margins and in the follow-up of patients in the outpatient setting while they receive antifungal therapy. Thus, the surgeon will be able to conservatively guide his/her debridement to the infected areas only and prevent further iatrogenic morbidity from the procedure. The very high PPV for all subsets suggests that any patient with a positive frozen-section result should be taken promptly to the operating room for surgical debridement. This would prevent the high rate of morbidity and mortality associated
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Otolaryngology–Head and Neck Surgery, Vol 136, No 5, May 2007
Figure 1 Mucor. (A) Frozen-section slide, hematoxylin and eosin stain. Broad, hyaline, thin-walled, mostly aseptate hyphae are observed. The shape of the hyphae is typically nonparallel, branch irregularly, and irregular in size. Note the organisms are seen in a background of necrosis (500⫻). (B) Permanent section slide, Gomori methanamine silver stain. This special stain is useful for the detection of fungi. This slide shows diffusely positively staining fungal organisms (500⫻).
with a delayed treatment that may occur if the physician is waiting for the lengthy process of permanent section. Given the lower NPV compared with PPV in this series, the authors believe it is advisable that multiple biopsies be taken to ensure accurate diagnosis. With an overall NPV of 72%, if a specimen is returned negative yet the clinical suspicion of invasive disease is high, it would be useful to continue to examine the patient in the operating room. In this manner, samples from multiple biopsy sites can be obtained for frozen-section and could confirm the suspicion of disease. A search of the relevant literature revealed a few small population studies and case reports in reference to the utility of frozen-section in the diagnosis of AIFRS. Hofman et al6 performed a retrospective study at their institution on a total of six patients. Their study came to the conclusion that frozen-section is sensitive and specific for early diagnosis of invasive mucormycosis. However, their patient population included six patients, all with invasive Mucor and none with invasive Aspergillus, and did not include numerical data to support their conclusions. Other case reports have described the use of frozen-section in the intraoperative setting. These reports described the use of frozen section as a tool to guide their procedures as a means to ensure thorough surgical debridement. The frozen-sections were also able to prevent total exenteration in the operating room by conserving those tissues that maintained negative margins.6-8 It is also reported, however, that frozen-section has limitations in its usefulness. In a recent case study by Adelson et al,9 a series of frozen-sections were sent during intraoperative debridement of a patient with AIFRS. All of the frozen-sections were reported to be negative for fungal elements. Later, however, they were found to be positive for Mucor on permanent section. This limitation of frozensection seems to be primarily in regards to Mucor species. It was observed in our own patient population that nearly all of our false negatives were in patients that were later diagnosed with Mucor on permanent section. This discrepancy
is likely because of the fact that Mucor has a less organized structure with more wide and loose forms, making it harder to visualize on frozen-section without special staining, whereas Aspergillus is more organized and tends to form fungal “balls” making it more readily identifiable.3 Alternative means of diagnosis have been used in the past, the most prominent of which has been the use of fungal cultures to show the presence of fungal species in patients suspected to have AIFRS. However, cultures remain an insensitive method to show their presence. Our study showed that fungal cultures had a sensitivity of only 54%. This held true regardless of the species grown, Mucor or Aspergillus. The slow nature of fungal growth further limits the utility of culture as a diagnostic tool in a disease process that is rapidly progressive. The benefit that culture has over frozen-section is that it allows speciation of the invasive fungus and determines antifungal sensitivity to direct medical therapy.3,13 In addition, there have been a few, selected case reports of the use of fine-needle aspiration cytology in showing the existence of fungal species in patients with suspected AIFRS. This method has been shown within these reports to be reliable; however, the data have been limited in the literature so far.14,15 In addition, even if fungal species are found on either the culture or on the fine-needle aspirate, they only show the presence of the fungi. There can be no definitive means to prove that these fungi have actually become invasive in the patient using these methods. Despite aggressive treatment by (1) reversing the underlying immunosuppression, (2) high dose antifungal therapy, and (3) aggressive surgical debridement, the prognosis of this disease remains generally quite poor.1,2,12,13,16 Survival rates have increased to near 80% among diabetics, largely because of the improvements in treatment of diabetic ketoacidosis combined with early clinical detection and management of fungal disease.2 However, survival rates are still as low as 20% to 50% in those patients in whom the underlying immunosuppression is not as easily corrected
Ghadiali et al
Frozen-section biopsy analysis for acute invasive . . .
719
Figure 2 Aspergillus. (A) Frozen-section slide, hematoxylin and eosin stain. Hyaline, septate, branched hyphae of uniform diameter are observed. The branching is characteristically at a 45° angle (arrow). Organisms are present in a background of necrosis (500⫻). (B) Permanent section slide, Aspergillus stain (immunoperoxidase). This slide shows diffusely positively staining fungal organisms (500⫻).
(eg, in leukemic patients).1 This raises the point that the astute clinician must entertain the possibility of a diagnosis of AIFRS in all immunocompromised patients with sinus complaints despite an innocuous appearance at the bedside or on CT scan. Frozen-section has been shown in this study to be a highly predictive diagnostic tool in the diagnosis of AIFRS in the event that the patient’s clinical appearance alone does not indicate invasive disease. It is useful for both invasive Mucor and invasive Aspergillus and for both bedside diagnosis and surgical debridement.
CONCLUSION In our study, we have shown that frozen-section biopsy is a highly predictive tool for a rapid and effective diagnosis in patients with suspected acute invasive fungal rhinosinusitis for both invasive mucormycosis and invasive aspergillosis. Microbiological cultures, although useful for mycological speciation, are less sensitive. Furthermore, we have shown that frozen-section has a strong role in the operating room as a tool to guide surgical debridement and/or antifungal therapy. Permanent pathology section remains the gold standard for the diagnosis of invasive fungal sinusitis.
Acknowledgment We appreciate the valuable assistance provided by Daniel Kinnamon, MS, for the biostatistical analysis in this study.
REFERENCES 1. Ferguson B. Mucormycosis of the nose and paranasal sinuses. Otolaryngol Clin North Am 2000;33:349 – 65.
2. Dhong H, Lanza D. Fungal rhinosinusitis. In: Kennedy D, Zinreich J, Bolger W, editors. Diseases of the sinuses. Cambridge: Blackwell Science, Inc. 2001. p. 179 –95 3. Frater J, Hall G, Procop G. Histologic features of zygomycosis: emphasis on perineural invasion and fungal morphology. Arch Pathol Lab Med 2001;125:375– 8. 4. Clancy C, Nguyen M. Invasive aspergillosis in apparently immunocompetent hosts. J Infect 1998;37:229 – 40. 5. Dhiwakar M, Thakar A, Bahadur S. Improving outcomes in rhinocerebral mucormycosis—Early diagnostic pointers and prognostic factors. J Laryngol Otol 2003;117:861–5. 6. Hofman V, Castillo L, Betis F, et al. Usefulness of frozen section in rhinocerebral mucormycosis diagnosis and management. Pathology 2003;35:212– 6. 7. Langford J, McCartney D, Wang R. Frozen section-guided surgical debridement for management of rhino-orbital mucormycosis. Am J Ophthalmol 1997;124:265–7. 8. Pelton R, Peterson E, Patel B, et al. Successful treatment of rhinoorbital mucormycosis without exenteration. Ophthal Plast Reconstr Surg 2001;17:62– 6. 9. Adelson R, Zito J, Romaguera R. Invasive fungal adenoiditis. Otolaryngol Head Neck Surg 2006;134:713– 4. 10. Liang K, Zeger S. Longitudinal data analysis using generalized linear models. Biometrika 1986;73:13–22. 11. Leemis L, Trivedi K. A comparison of approximate interval estimators for the Bernoulli parameter. Am Stat 1996;50:63– 8. 12. Strasser M, Kennedy R, Adam R. Rhinocerebral mucormycosis. Therapy with amphotericin B lipid complex. Arch Intern Med 1996;156: 337–9. 13. Peterson K, Wang M, Canalis R, et al. Rhinocerebral mucormycosis: evolution of the disease and treatment options. Laryngoscope 1997; 107:855– 62. 14. Nair M, Kapila K, Verma K. Fine needle aspiration: another diagnostic modality for rhinocerebral mucormycosis. Diagn Cytopathol 1999;21: 300 –1. 15. Deshpande A, Munshi M. Rhinocerebral mucormycosis: diagnosis by aspiration cytology. Diagn Cytopathol 2000;23:97–100. 16. Gupta A, Mann S, Khosla V, et al. Non-randomized comparison of surgical modalities for paranasal sinus mycoses with intracranial extension. Mycoses 1999;42:225–30.
ORIGINAL RESEARCH
Frozen-section biopsy analysis for acute invasive fungal rhinosinusitis Murtaza T. Ghadiali, MD, Nathan A. Deckard, BS, Uzma Farooq, MD, Frank Astor, MD, Phillip Robinson, MD, and Roy R. Casiano, MD, Miami, FL OBJECTIVE: To evaluate sensitivity and specificity of frozensection biopsy in the diagnosis of acute invasive fungal rhinosinusitis (AIFRS). STUDY DESIGN AND SETTING: Retrospective chart review of all patients treated for AIFRS at the University of Miami between 1993 and 2005. Twenty patients with the clinical diagnosis of AIFRS were identified. Histopathological data were collected to evaluate the use of frozen-section biopsy to diagnose the disease. RESULTS: Permanent pathology sections were positive for AIFRS in all specimens. Frozen-section biopsies were positive for fungal features in all but five cases (four with Mucor and one with Aspergillus). High positive predictive values were found for both fungal types and for both sets of patients (bedside and intraoperative). CONCLUSION: Frozen-section biopsy is a useful tool for rapid and effective diagnosis in patients with suspected AIFRS. A positive frozen-section result is highly predictive of invasive disease and merits prompt surgical intervention. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
A
cute invasive fungal rhinosinusitis (AIFRS) is an uncommon, opportunistic infection seen primarily in diabetic and immunocompromised patients. The infections are predominantly by species from the order Mucorales (including Absidia, Mucor, Rhizomucor, and Rhizopus) and by Aspergillus. The spores of these fungi are ubiquitous in the environment. As a result, exposure is common in all population groups, although progression to invasion and infection is usually avoided with an intact immune system.1,2
From the University of Miami/Jackson Memorial Hospital, Department of Otolaryngology/Head and Neck Surgery (Drs Ghadiali, Astor and Casiano), Department of Pathology (Drs Farooq and Robinson) and the University of Miami Miller School of Medicine (N. Deckard). Disclosure: Dr Caslano: Gyrus ENT-royalties, consultant; Medtronics. Presented at the American Academy of Otolaryngology–Head and Neck Surgery, Toronto, Canada, September 19, 2006.
Mucormycosis in particular has been associated primarily with diabetes mellitus patients in ketoacidosis.2 Other patient groups commonly affected by AIFRS include those with neutropenia, immunological disorders, chemotherapy, and transplant recipients. The progression of the disease depends on the severity of immunosuppression; the greater the immunocompromise, the more rapid the progression.1 AIFRS first affects the turbinates and sinuses resulting in an acute sinusitis. The infection can then spread to contiguous structures such as the palate, the orbit, and the brain leading to cranial nerve palsies, invasion of the carotid artery, and mental obtundation, thus making early diagnosis key before such extensive invasion occurs.1 Mucor has a predilection for the internal elastic lamina of the arterial blood vessels and spreads by angioinvasion. This causes tissue necrosis as the arterial blood flow is occluded, resulting in infarction and the black eschars that are pathognomonic for the disease.2,3 Furthermore, it has been shown that perineural invasion is also an integral aspect of the disease process of Mucor and can be used to identify the invasive nature of this fungus on histological examination.3 Aspergillus has a similar picture in that, in its invasive form, its hyphae may also invade the vasculature and spread by angioinvasion. It may also spread directly across tissue planes.4 Although sinus CT is the preferred imaging modality to evaluate for signs of invasion by the disease, bony destruction is often seen only late in the course of the disease after soft-tissue necrosis has already occurred. AIFRS can present without any evidence of invasion on an essentially Reprint requests: Murtaza T. Ghadiali, MD, University of Miami Hospital and Clinics, c/o Dr Roy R. Casiano, 1475 NW 12th Avenue, Room 4025, Miami, FL 33136. E-mail address: [email protected].
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.01.002
Ghadiali et al
Frozen-section biopsy analysis for acute invasive . . .
normal CT scan.5 Therefore, any clinical indication of an invasive process warrants a biopsy at the bedside or in the operating room to confirm a tissue diagnosis of invasive fungal disease. Frozen-sections at the time of the original biopsy have been recommended to obtain the earlier diagnosis so that medical treatment can begin as soon as possible in the event of a positive diagnosis.2,6-9 Past studies have found frozen-section to be useful in diagnosis and even during surgical debridement.6-8 However, there is some evidence that its usefulness is limited.9 There is a paucity of reports in the literature that have statistically evaluated frozen-section as a diagnostic tool in the evaluation of AIFRS patients. In this study, we have conducted a retrospective review of patients’ charts who have undergone surgery for this diagnosis, looking specifically at the predictive value of frozen-section to diagnose the condition.
METHODS The medical records from the University of Miami/Jackson Memorial Hospital were reviewed from 1993 to 2005 for all cases of acute invasive fungal rhinosinusitis after approval was obtained from the institutional review board. All cases of invasive Mucor and Aspergillus infections proven by permanent section that also had frozen-sections performed were included in this retrospective chart review. Data on age, sex, comorbidities, and clinical presentation were collected from the charts. Specific comorbidities that were noted were those that cause an immunocompromised state, such as diabetes mellitus, solid-organ transplant, immune system disorders, neutropenia, chemotherapy, and HIV/AIDS. Frozen-section slides were stained with hematoxylin and eosin. Permanent sections were stained with Gomori methanamine silver and immunoperoxidase antibody stains. All slides were analyzed under the microscope at 500⫻ by the University of Miami/Jackson Memorial Hospital Department of Pathology. Mucor was identified by its broad, thin-walled, and sparsely septated hyphae. The hyphae are typically nonparallel, branch irregularly, and are irregular in size (Fig 1). Aspergillus was identified by its septated and regularly branched hyphae of uniform diameter. The branching is characteristically at a 45° angle (Fig 2). Sinus tissue was sent to the microbiology laboratory and cultured using Inhibitory Mold Agar and Sabouraud Dextrose Agar per laboratory protocol. Histopathological examination reports (including frozen and permanent section specimens used to diagnose patients before treatment, during intraoperative debridement, and postoperatively) as well as microbiological culture results were obtained and analyzed.
STATISTICAL ANALYSIS A generalized estimating equation (GEE) approach10 with a logit link and a working independence correlation structure
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was used when estimating 95% confidence intervals for sensitivity and negative predictive value (NPV) to take into account potential correlations between outcomes in samples from several sites in the same individuals. The GEE technique adjusts the standard errors to provide appropriate asymptotic inferences in repeated measures data. For specificity and positive predictive value (PPV), exact Blyth 95% confidence intervals11 were calculated because these quantities were 1, meaning that there would have been numerical problems with GEE estimates. Assuming positive correlations between the outcomes in samples on the same individual, the true confidence intervals would probably be wider. Differences in the sensitivity and NPV between different fungal species as well as intraoperative and other patients were tested for significance by entering a group main effect into the intercept-only GEE model and assessing the P value from the Wald test. These models also yielded 95% confidence intervals for sensitivity and negative predictive value in these different groups. Because specificity and PPV were both 1 in all groups, we could not perform any tests for differences in these quantities between different fungal species and intraoperative and other patients. However, exact Blyth 95% confidence intervals for these quantities were calculated in these subgroups. All tests were 2 sided, and alpha was set at 0.05. All analyses were performed by using SAS/STAT software, version 9.1.3 of the SAS System for Windows (SAS Institute Inc, Cary, NC).
RESULTS A total of 20 patients with the clinical diagnosis confirmed by histopathology on permanent section that also had frozensections performed were identified from a chart review from 1993 to 2005. The age range was from 8 to 76 years, with an average age of 44 years. There were a total of 9 males and 11 females (Table 1). The study included 11 patients with a diagnosis of invasive mucormycosis and 9 patients with invasive aspergillosis. Of these, the most common predisposing illness was diabetes mellitus, occurring in 11 patients (55%). Solidorgan transplant–related immunocompromise was found in five patients (25%), HIV/AIDS was a diagnosis in five patients (25%), and leukemia was a diagnosis in one patient (5%). Two patients had no identified immunocompromised states that would predispose them to invasive fungal disease. Some of the patients in the study had multiple comorbidities. The most common presenting signs and symptoms included facial pain and swelling in 9 of 20 patients (45%), proptosis/visual changes in 8 of 20 patients (40%) and headaches in 4 of 20 patients (20%). Other presenting symptoms included sinusitis, bloody nasal drainage/epistaxis, necrotic hard palate, and V2 numbness. Among the 20 patients, a total of 44 samples were collected that were processed for both frozen and permanent
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Table 1 Frozen-section Patient
Age
Sex
Comorbidities
SSx
Dx
1 2 3
50 61 76
F M F
HIV/AIDS, DM KTSPL, DM DM
Vision loss, proptosis Facial pain Facial pain, swelling
Mucor Mucor Aspergillus
4 5 6
63 35 17
M F F
DM LivTSPL DM
Facial pain, HA Necrotic palate Facial pain, swelling
Mucor Mucor Mucor
7 8 9 10 11
49 35 31 59 72
F F M M F
LuTSPL, DM HIV/AIDS HIV/AIDS HIV/AIDS DM
Facial pain, swelling Vision loss, proptosis Facial swelling, proptosis Blindness, HA Facial swelling, proptosis
Aspergillus Aspergillus Aspergillus Aspergillus Mucor
12 13 14 15 16 17
43 40 8 25 38 48
M M F M F F
None Acute Leukemia KTSPL, DM N/A DM DM
Sphenoid sinusitis Bilateral sinusitis Facial pain, swelling Nasal mass, sinusitis Vision loss Facial swelling
Aspergillus Mucor Mucor Aspergillus Mucor Mucor
18 19 20
21 62 49
F M M
HIV/AIDS DM LivTSPL
Vision loss, epistaxis HA, EOM palsy HA, V2 numbness
Aspergillus Aspergillus Mucor
Frozensection
Permanent section
Positive ⫻1 Positive ⫻1 Positive ⫻1 Negative ⫻7 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻7 Negative ⫻1 Negative ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻2
Positive ⫻1 Positive ⫻1 Positive ⫻1 Negative ⫻7 Positive ⫻1 Positive ⫻1 Positive ⫻2 Positive ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻7 Positive ⫻1 Negative ⫻1 Positive ⫻1 Positive ⫻1 Positive ⫻2 Positive ⫻1 Positive ⫻1 Positive ⫻2 Negative ⫻1 Negative ⫻1 Negative ⫻1 Positive ⫻2 Negative ⫻2
DM, diabetes mellitus; KTSPL, kidney transplant; LivTSPL, liver transplant; LuTSPL, lung transplant; HA, headache; EOM, extraocular muscle *Italicized entries denote specimens obtained during intraoperative debridement.
section. All 20 cases were positive on permanent section for invasive fungal disease. For patients 18 and 19, the biopsies sent for both frozen-section and permanent section were returned as negative as noted in Table 1. However, separate biopsies were sent for permanent section only, which confirmed the diagnosis of AIFRS. Frozen-section results were positive for fungal features in all but 5 of the 44 samples collected. Four biopsies with Mucor and one with Aspergillus were falsely negative. Using permanent section as the gold standard, overall sensitivity for frozen-section was 0.84 (95% confidence interval [CI], 0.67-0.93), specificity was 1 (95% CI, 0.75-1), NPV was 0.72 (95% CI, 0.37-0.92), and PPV was 1 (95% CI, 0.87-1). For Mucor alone, sensitivity was 0.83 (95% CI, 0.64-0.93), specificity was 1 (95% CI, 0.39-1), NPV was 0.5 (95% CI, 0.18-0.82), and PPV was 1 (95% CI, 0.83-1). For Aspergillus alone, sensitivity was 0.86 (95% CI, 0.42-0.98), specificity was 1 (95% CI, 0.66-1), NPV was 0.90 (95% CI, 0.42-0.99), and PPV was 1 (95% CI, 0.54-1). No statistical difference was found for the sensitivity or NPV of frozen-section biopsy between the two fungal groups (P ⫽ 0.88 and 0.14, respectively). However, given the large confidence intervals for these figures, the study may not have been adequately powered.
Six of the 20 patients had frozen-sections performed intraoperatively to guide the surgical debridement. A total of 30 slides were performed on these six patients alone. Within this group, the sensitivity for frozen-section was 0.84 (95% CI, 0.62-0.94), specificity was 1 (95% CI, 0.72-1), NPV was 0.79 (95% CI, 0.35-0.96), and PPV was 1 (95% CI, 0.79-1). For the nonintraoperative group, the sensitivity for frozen-section was 0.83 (95% CI, 0.52-0.96) and NPV was 0.5 (95% CI, 0.120.88). No statistical difference was found for the sensitivity or NPV of frozen-section biopsy between the two groups, intraoperative and nonintraoperative patients (P ⫽ 0.95 and 0.35, respectively). However, again, the large confidence intervals for these data may indicate an inadequate power for this study (Table 2). Using permanent section as the gold standard, overall culture results were positive in only 11 of the 20 cases. Overall sensitivity for cultures was 0.55 (95% CI, 0.340.74). For Mucor alone, sensitivity was 0.54 (95% CI, 0.29-0.78). For Aspergillus alone, sensitivity was 0.55 (95% CI, 0.27-0.80). Specificity was not calculated for cultures because there were neither true negatives nor false positives in our population (Table 3).
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Table 2 Intraoperative frozen-section Patient
# sent intraop
FS Diagnosis
#TP
#FP
#TN
#FN
PS diagnosis
3 6 11 14 17 20 Totals:
8 4 9 2 3 4 30
Aspergillus Mucor Mucor Mucor Mucor Mucor
1 2 7 2 2 2 16
0 0 0 0 0 0 0
7 0 1 0 1 2 11
0 2 1 0 0 0 3
Aspergillus Mucor Mucor Mucor Mucor Mucor
DISCUSSION Acute invasive fungal rhinosinusitis is an uncommon infection that occurs almost exclusively in immunocompromised patients. An early diagnosis allows the prompt institution of medical and surgical treatment in order to avoid the high morbidity and mortality associated with the progression of the disease.1,2,12,13 The problem that clinicians face in practice is finding a rapid and reliable means by which to diagnose the infection. The gold standard of diagnosis is pathological examination on permanent sections.1-3 However, permanent section can be a lengthy and time-consuming means to obtain the diagnosis, delaying the appropriate treatment and increasing the morbidity and mortality of the disease. The current literature available on the accuracy and utility of frozensection in the diagnosis of this disease has been sparse. The literature has thus far been limited in scope to case reports and one small retrospective study that described the use of frozen-section in diagnosis but included only Mucor pa-
Table 3 Culture results Patient
PS diagnosis
Culture diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Mucor Mucor Aspergillus Mucor Mucor Mucor Aspergillus Aspergillus Aspergillus Aspergillus Mucor Aspergillus Mucor Mucor Aspergillus Mucor Mucor Aspergillus Aspergillus Mucor
Negative Rhizopus A. flavus Rhizopus Negative Negative A. fumigatus A. fumigatus A. fumigatus A. fumigatus Rhizopus Negative Negative Rhizopus Negative Rhizopus Negative Negative Negative Rhizopus
tients and did not convey any numerical data for the sensitivity and specificity of the procedure.6-9 This study differs from the existing literature in that our patient population contains a total of 44 frozen-section specimens, includes both Mucor and Aspergillus in the analysis, and provides statistical analyses to support its conclusions. In this retrospective study, a total of 44 frozen-sections were obtained from 20 patients. The utility of frozen-section biopsy was evaluated on a site-specific basis to compare how well it performs as compared with permanent section. Only five were found to be false negatives based on the final permanent section results of the same specimens, showing a sensitivity of 84% and an NPV of 72%. In addition, the narrow confidence interval of the PPV shows that frozensection biopsy is highly predictive of invasive disease. Therefore, frozen-section diagnosis was found to be a useful tool for the early diagnosis of AIFRS in the event that the patients’ clinical appearance alone does not indicate invasive disease to the examiner. Furthermore, no statistical difference was found for the sensitivity or the NPV of frozen-section between the two most common fungal species to cause AIFRS, Mucor and Aspergillus. Frozen-section does not just provide an early diagnosis in the clinical setting. It can also provide useful information for frozen-section– guided intraoperative and postoperative debridement in the operating room. Six of the patients in our review used this method as a part of their surgical treatment. A total of 30 slides were obtained within this group alone. Using the pathological reports, it was found that intraoperative frozen-section results for sensitivity and NPV were not statistically significant from the nonintraoperative patients. This is useful to the surgeon in guiding his/her procedure. By knowing that frozen-section is a reliable tool, it can be used to prevent more extensive surgical debridement by providing intraoperative negative margins and in the follow-up of patients in the outpatient setting while they receive antifungal therapy. Thus, the surgeon will be able to conservatively guide his/her debridement to the infected areas only and prevent further iatrogenic morbidity from the procedure. The very high PPV for all subsets suggests that any patient with a positive frozen-section result should be taken promptly to the operating room for surgical debridement. This would prevent the high rate of morbidity and mortality associated
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Figure 1 Mucor. (A) Frozen-section slide, hematoxylin and eosin stain. Broad, hyaline, thin-walled, mostly aseptate hyphae are observed. The shape of the hyphae is typically nonparallel, branch irregularly, and irregular in size. Note the organisms are seen in a background of necrosis (500⫻). (B) Permanent section slide, Gomori methanamine silver stain. This special stain is useful for the detection of fungi. This slide shows diffusely positively staining fungal organisms (500⫻).
with a delayed treatment that may occur if the physician is waiting for the lengthy process of permanent section. Given the lower NPV compared with PPV in this series, the authors believe it is advisable that multiple biopsies be taken to ensure accurate diagnosis. With an overall NPV of 72%, if a specimen is returned negative yet the clinical suspicion of invasive disease is high, it would be useful to continue to examine the patient in the operating room. In this manner, samples from multiple biopsy sites can be obtained for frozen-section and could confirm the suspicion of disease. A search of the relevant literature revealed a few small population studies and case reports in reference to the utility of frozen-section in the diagnosis of AIFRS. Hofman et al6 performed a retrospective study at their institution on a total of six patients. Their study came to the conclusion that frozen-section is sensitive and specific for early diagnosis of invasive mucormycosis. However, their patient population included six patients, all with invasive Mucor and none with invasive Aspergillus, and did not include numerical data to support their conclusions. Other case reports have described the use of frozen-section in the intraoperative setting. These reports described the use of frozen section as a tool to guide their procedures as a means to ensure thorough surgical debridement. The frozen-sections were also able to prevent total exenteration in the operating room by conserving those tissues that maintained negative margins.6-8 It is also reported, however, that frozen-section has limitations in its usefulness. In a recent case study by Adelson et al,9 a series of frozen-sections were sent during intraoperative debridement of a patient with AIFRS. All of the frozen-sections were reported to be negative for fungal elements. Later, however, they were found to be positive for Mucor on permanent section. This limitation of frozensection seems to be primarily in regards to Mucor species. It was observed in our own patient population that nearly all of our false negatives were in patients that were later diagnosed with Mucor on permanent section. This discrepancy
is likely because of the fact that Mucor has a less organized structure with more wide and loose forms, making it harder to visualize on frozen-section without special staining, whereas Aspergillus is more organized and tends to form fungal “balls” making it more readily identifiable.3 Alternative means of diagnosis have been used in the past, the most prominent of which has been the use of fungal cultures to show the presence of fungal species in patients suspected to have AIFRS. However, cultures remain an insensitive method to show their presence. Our study showed that fungal cultures had a sensitivity of only 54%. This held true regardless of the species grown, Mucor or Aspergillus. The slow nature of fungal growth further limits the utility of culture as a diagnostic tool in a disease process that is rapidly progressive. The benefit that culture has over frozen-section is that it allows speciation of the invasive fungus and determines antifungal sensitivity to direct medical therapy.3,13 In addition, there have been a few, selected case reports of the use of fine-needle aspiration cytology in showing the existence of fungal species in patients with suspected AIFRS. This method has been shown within these reports to be reliable; however, the data have been limited in the literature so far.14,15 In addition, even if fungal species are found on either the culture or on the fine-needle aspirate, they only show the presence of the fungi. There can be no definitive means to prove that these fungi have actually become invasive in the patient using these methods. Despite aggressive treatment by (1) reversing the underlying immunosuppression, (2) high dose antifungal therapy, and (3) aggressive surgical debridement, the prognosis of this disease remains generally quite poor.1,2,12,13,16 Survival rates have increased to near 80% among diabetics, largely because of the improvements in treatment of diabetic ketoacidosis combined with early clinical detection and management of fungal disease.2 However, survival rates are still as low as 20% to 50% in those patients in whom the underlying immunosuppression is not as easily corrected
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Figure 2 Aspergillus. (A) Frozen-section slide, hematoxylin and eosin stain. Hyaline, septate, branched hyphae of uniform diameter are observed. The branching is characteristically at a 45° angle (arrow). Organisms are present in a background of necrosis (500⫻). (B) Permanent section slide, Aspergillus stain (immunoperoxidase). This slide shows diffusely positively staining fungal organisms (500⫻).
(eg, in leukemic patients).1 This raises the point that the astute clinician must entertain the possibility of a diagnosis of AIFRS in all immunocompromised patients with sinus complaints despite an innocuous appearance at the bedside or on CT scan. Frozen-section has been shown in this study to be a highly predictive diagnostic tool in the diagnosis of AIFRS in the event that the patient’s clinical appearance alone does not indicate invasive disease. It is useful for both invasive Mucor and invasive Aspergillus and for both bedside diagnosis and surgical debridement.
CONCLUSION In our study, we have shown that frozen-section biopsy is a highly predictive tool for a rapid and effective diagnosis in patients with suspected acute invasive fungal rhinosinusitis for both invasive mucormycosis and invasive aspergillosis. Microbiological cultures, although useful for mycological speciation, are less sensitive. Furthermore, we have shown that frozen-section has a strong role in the operating room as a tool to guide surgical debridement and/or antifungal therapy. Permanent pathology section remains the gold standard for the diagnosis of invasive fungal sinusitis.
Acknowledgment We appreciate the valuable assistance provided by Daniel Kinnamon, MS, for the biostatistical analysis in this study.
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